Nanoliposomal vaccine containing long multi-epitope peptide E75-AE36 pulsed PADRE-induced effective immune response in mice TUBO model of breast cancer

Highlights

• Long peptide induced effective immune response in mice TUBO model of breast cancer.

• Addition of PADRE led to an improvement of immunogenic peptide based cancer vaccines.

• Liposomal formulation containing antigenic peptides significantly induced stronger CD8⁺ T cell immune responses.

Abstract

The main goal of peptide-based cancer vaccines is to induce the immune system and activation of effective T cell responses against cancerous cells. Nevertheless, the potency of peptide vaccines is insufficient in most of cases and had limited clinical success. Therefore, the optimization of peptide-based cancer vaccine is essential to achieve powerful therapeutic outcomes. One strategy to enhanced potency of peptide vaccines and induce strong immune responses is the preparation of multi-epitope peptide formulation containing both Th- and cytotoxic T lymphocyte–induced responses epitope using suitable delivery system. For this reason, we studied the effect of Dioleoylphosphatidylethanolamine-containing liposomal vaccine composed of a mixture of short peptides AE36 and E75 (HER2/neu-derived peptides) and long multi-epitope peptide E75-AE36 (linkage of short peptides) in combination with a Pan HLA-DR epitope (PADRE) peptide. These formulations were examined using a series of subcutaneously injection to HER-2⁺ TUBO-tumoured mice in prophylactic and therapeutic model. We observed that mice vaccinated with liposomal long peptide in combination with PADRE resulted in the superior induction of CD4⁺ and CD8⁺ T cells responses and significantly enhanced production of IFN-γ compared with liposomal short peptides and non-liposomal peptides formulations. Moreover, liposome-long peptide with PADRE led to the considerable reduction of tumour growth and lifespan induction in mouse model. In conclusion, our study indicated that liposomal formulation containing long multi-epitope peptide E75-AE36 with PADRE could be used as an effective multi-epitope prophylactic/therapeutic vaccine to generate potent antigen-specific CD8⁺ T-cell immune response and may be introduced as a possible candidate peptide vaccine for breast cancer.

Introduction

The main goal of the peptide-based cancer vaccines is to induce the host immune system to recognize and eradicate the target malignant cells using a defined set of antigens especially tumour-associated antigens (TAAs) [1]. Nevertheless, most of the developing peptide-based cancer vaccines have limited success in clinical study with rather poor immunological responses. Therefore, the optimization of the peptide-based vaccine is essential to achieve powerful therapeutic outcomes.

To date, various strategies have been exploited to improve the potency of peptide-based vaccines, including use of long multi-epitope peptides containing optimum cytotoxic T lymphocyte (CTL) and Th epitopes of TAA, MHC class I and II epitope hybrid vaccines, simultaneous stimulation of CD8⁺ cytotoxic and CD4⁺ T helper lymphocytes, peptide pulsed dendritic cells (DCs) therapy, using suitable delivery system and adjuvants [[2], [3], [4]].

It is reported that when a peptide, as a vaccine, was administered subcutaneously, peptides (especially short peptide with 9–10 amino acids length) may be rapidly cleared from the site of injection or may bind to non-professional antigen-presenting cells (APCs), such as fibroblasts that leads to the development of immune tolerance. In addition, it is possible that the peptide rapidly degraded by different peptidases and resulted in poor peptide/MHC presentation. In addition, some antigenic peptides with endogenous origin, such as peptides derived from TAAs, have poor immunogenicity and not effectively recognized by immune system [2]. Therefore, it seems that this peptide is not effective to produce a long-term CTL immune response [5]. In contrast to short peptides, synthetic long peptides (25–50 amino acids) containing optimum CTL and Th epitopes induced a stronger immune response and markedly improved antigen-specific CTL responses, owing to long peptide firstly taken up by professional APC such as DCs and undergo intracellular processing and then presented to the MHC molecules. in addition, cross-presentation mechanism led to effective activation of CD4⁺ and CD8⁺ T cells and inducing stronger immunity rather than tolerance [[6], [7], [8], [9]]. Hence, it seems that the design of long multiple epitopes peptide vaccine could enhance the efficacy of peptide vaccine and may elicit strong and long-lived CTL responses.

On the other hand, one of the well-known strategy to promote vaccine potency is the preparation of a particulate system containing both antigenic epitopes for Th and CTLs stimulations. The potential advantage of these strategies lies in increased chance for simultaneous presentation of both MHC class I and class II epitopes on the surface of a single APC, thereby facilitating concurrent induction of CD4⁺ and CD8⁺ T cells and subsequently activation of both Th and CTL responses. It is shown that CD4⁺ T helper cells are necessary for the effective induction of a robust CD8⁺ CTL response and expansion of memory CD8⁺ T cells [[10], [11], [12]]. Furthermore, CD4⁺ T cells are especially important for the enhanced activation of DCs through increasing expression of costimulatory molecules and their antigen presentation [10]. Thus, it is desirable to design vaccines containing both CD4⁺ T and CD8⁺ T cells specific epitopes to induce both CD4⁺ and CD8⁺ T cells responses and enhance the vaccine potency. Pan HLA-DR epitope (PADRE) is DR-restricted T helper epitopes and is one of the most effective molecules used to induce CD4⁺ T responses. Recent studies indicated that PADRE acts as a potent adjuvant and capable to enhance vaccine potency [13,14]. In this regard, our previous study showed that addition of PADRE to the liposomal formulation enhanced the potency of peptide-based liposomal vaccines and markedly improved the production of IFN-γ and increased frequency of CD8⁺ T cells in animal model of breast cancer [3].

Moreover, extensive evidence showed that the use of an appropriate delivery system has crucial role in far more robust induction of immune response. Over the past few years, liposomes have attracted considerable interest as carriers for delivery of antigens and adjuvants in cancer immunotherapies [[15], [16], [17]]. Our previous studies have shown that liposomes are able to elicit strong immune responses [3,[18], [19], [20]].

Human epidermal growth factor receptor 2 (HER2/neu) is one of the most well-known TAAs that is overexpressed in 15–30% of invasive breast cancers [21]. Recent studies indicated that HER2-derived peptides are a remarkable target for peptide-based cancer vaccines that were able to induce and elicite a robust anti-HER2–specific immune responses in human and animal models. Over the years, various vaccines containing different HER2-derived peptides have been tested for their therapeutic efficacy in both human and animal studies [3,[22], [23], [24]]. Among different immunogenic peptide derived from the HER2 protein, most extensive studies have been performed on E75 and AE36 [25,26]. E75 (Nelipepimut-S) is a nine-amino acid peptide from the extracellular domain of HER2 protein (HER2/neu 369–377, KIFGSLAFL) emerged as an effective antigen for vaccination [26]. E75 could bind to HLA-A2 and HLA-A3 molecules and stimulate CTLs to detect and destroy HER2-expressing tumour cells [26]. Moreover, AE36, a 15 amino acid peptide from the intracellular domain of the HER2 protein (HER2/neu 776–790, Ac-GVGSPYVSRLLGICL-NH2) has been used as immunogenic peptide in several formulations. AE36 was predicted to bind to MHC class II molecules and was able to stimulate CD4⁺ Th and CD8⁺ T cytotoxic lymphocytes. Previously indicated that vaccination with the E75 and AE36 peptide in combination with GM-CSF could increase IFN-γ production and enhancement of cytotoxic T cell anti-tumour activity in patients with breast cancer [25,27].

Based on the importance of multi-epitope peptide vaccines and liposome-based delivery systems, we hypothesized that Dioleoylphosphatidylethanolamine (DOPE)-containing liposomal formulations carrying both AE36 and E75 in conjunction with PADRE may be more effective in induction of immune responses and may be introduced as a possible candidate for a potential breast cancer vaccine. To accomplish this, we developed a liposomal formulations composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG):Chol:DOPE containing both AE36 and E75 peptides in conjugation with PADRE peptide and then the immunogenicity and anti-tumour efficacy of them was evaluated. For this reason, we have designed two multi-epitope peptide-based formulation: (1) formulations containing a mixture of short length peptides AE36 and E75 (Short Peps) and (2) formulations containing long multi-epitope peptide E75-AE36 (Long Pep) in both liposomal and non-liposomal forms. The in vitro and in vivo studies were evaluated with prophylactic (vaccination) and therapeutic injections of the peptide formulation in TUBO breast cancer model of BALB/c mice.